Environmental modification of tolerance to morphine discriminative stimulus properties in rats

The development of tolerance to the discriminative stimulus properties of morphine was examined in rats trained to discriminate saline and 3.2 mg/kg morphine under amultiple timeout 15 min, 5 min fixed-ratio 30 schedule of food delivery. Generalization gradients were generated by administering increasing doses of morphine before successive timeout periods within the experimental session. Over the course of the study, the minimal discriminable dose (MDD) of morphine under control conditions fluctuated but did not systematically increase or decrease. Acute pretreatments of 3.2–17.8 mg/kg morphine 4–24 h before a generalization test resulted in minor changes in the MDD. To examine development of tolerance, supplemental doses of morphine (17.8 mg/kg) or saline were administered twice daily while discrimination training was either suspended or continued. Tolerance was assessed by weekly generalization tests. Greater tolerance developed to the morphine stimulus when training was suspended than when training was continued. For both training conditions, response rates during generalization tests were markedly suppressed during supplemental morphine administration, and original generalization gradients were recaptured within 2 weeks after termination of supplemental morphine administration. Supplemental saline administration did not alter the discriminative or rate-altering effects of morphine under either training condition. Thus, the magnitude of tolerance to a morphine discriminative stimulus reflected an interaction of supplemental drug treatment with the training conditions imposed during that treatment.     

Behavioral contingencies modulate tolerance to discriminative stimulus effects of morphine

Experiments examined how learning processes modulate tolerance to discriminative stimulus effects of morphine. Rats were trained to discriminate saline and 3.2 mg/kg morphine, and the doses of morphine required to mimic the training dose were determined before, during and after repeated treatment with saline or high doses of morphine (10 mg/kg, b.i.d.). In one set of experiments, training was either suspended or continued with saline and the original training dose during a 2-week treatment regimen. When training was suspended, high-dose morphine treatment increased the dose of morphine required for stimulus effects approximately 3-fold. Tolerance persisted 2 days after treatment ended, but disappeared within 7 days. In contrast, continued training with saline and 3.2 mg/kg morphine during high-dose treatment both attenuated development of tolerance and transferred control to lower doses. Transfer of control to lower doses appeared conditional upon recent termination of high-dose treatment, as it disappeared within 7 days. Treatment with saline did not change the doses of morphine required for stimulus effects under either training condition. A final experiment examined whether high-dose treatment could transfer control to higher doses of morphine. The treatment dose of 10 mg/kg morphine itself was used as the training dose during a 2-week treatment regimen. The dose of morphine required for stimulus effects increased 2- to 4-fold during treatment, but quickly returned to control values when treatment ended. These results extend previous findings that conditioning and pharmacodynamic processes jointly regulate development of tolerance to discriminative effects of morphine.     

Discriminative stimulus properties of valproic acid in the pigeon

Pigeons were successfully trained to discriminate 60 mg/kg valproic acid from saline using a two-key drug discrimination procedure. When 5–80 mg/kg doses of valproic acid were administered during generalization tests the percentage of responses directed to the valproic acid-appro-priate key varied directly with dose. the effects of administering the training dose of valproic acid at presession injection intervals ranging from 15 to 120 min were described by an inverted U-shaped function; the 30-min interval used during discrimination training engendered the largest percentage of valproic acid-appropriate responses. The discriminative stimulus properties of valproic acid failed to generalize to the anticonvulsant compounds phenobarbital (10, 20 mg/kg), phenytoin (2.5, 5 mg/kg), and ethosuximide (40, 80 mg/kg), indicating that not all anticonvulsant compounds share similar discriminative properties. Clonazepam (0.25, 0.50 mg/kg) and diazepam (1, 2 mg/kg), two benzodiazepines with anticonvulsant properties, produced quite different effects. The stimulus properties of valproic acid generalized to all doses of clonazepam, whereas intermediate generalization was evident with diazepam. Pentylenetetrazol (10, 20 mg/kg), chlorpromazine (5, 10 mg/kg), tripelennamine (2.5, 5.0 mg/kg), d-amphetamine (0.5, 1.0 mg/kg), morphine (1.25, 2.50 mg/kg), and imipramine (2.5, 5.0 mg/kg) induced only saline-like patterns of responding. The concomitant administration of pentylenetetrazol failed to antagonize the discriminative stimulus properties exerted by the training dose of valproic acid.     

Discrimination of a drug mixture in rats: role of training dose, and specificity

Many drugs produce compound discriminative stimuli with at least two elements; in contrast, the present study examines discrimination of a mixture of two drugs and tests the role of training dose in, and the specificity of, such a discrimination. Rats discriminated a mixture of nicotine (0.2mg/kg s.c.) and midazolam (0.1mg/kg s.c.) from saline in a two-bar operant conditioning procedure with accuracy of at least 80%. Stimulus control was analyzed by testing each drug separately. Initially, stimulus control was mainly attributable to the midazolam. The doses of drugs used to maintain the discrimination were then altered. As the training dose of nicotine increased and that of midazolam decreased, the magnitudes of responses to the separate drugs were progressively reversed, until stimulus control was mainly attributable to nicotine. Thus, responses to the components of the compound stimulus were systematically related to the amounts of drugs in the mixtures used to maintain the discrimination, and there was some evidence that a strong stimulus produced by one drug may have overshadowed a weaker stimulus produced by a different agent. To test specificity, generalization to other drugs was examined. There was no generalization to amphetamine, morphine or quipazine, up to doses that reduced overall rates of responding. It follows that cues produced by mixtures of drugs may be as specific as those produced by single agents.     

Discriminative stimulus effects of a nicotine-midazolam mixture in rats

Rats were trained to discriminate the effects of nicotine (0.4 mg/kg SC) plus midazolam (0.2 mg/kg SC) from those of saline in a two-bar operant conditioning procedure involving a tandem schedule of food reinforcement. After discrimination training, the component drugs of the mixture produced very considerable amounts of drug-appropriate responding when given separately. Mecamylamine and Ro 15-1788 only slightly attenuated the discriminative response to the mixture when given separately, but completely blocked the response when administered together. In different groups of rats trained to discriminate nicotine or midazolam separately from saline, neither drug appreciably altered the dose-response curve for the other, suggesting a minimal role for pharmacological interactions when effects of mixtures were assessed. The results suggest that the two components of a compound drug-produced stimulus can be perceived separately rather than being blended into a homogenous entity. Knowledge of the characteristics of compound drug-produced stimuli may aid interpretation of the discriminative effects of single drugs with wide spectra of action.     

Sensitization to the morphine-like discriminative stimulus effects of buprenorphine in rats.

An experiment was performed to determine whether chronic non-contingent administration of morphine would produce cross-sensitization to the cueing properties of buprenorphine or D-amphetamine. To this end the sensitivity to the discriminative stimulus effects of morphine, buprenorphine and D-amphetamine was determined in rats trained to discriminate 10 mg kg(-1)morphine from saline in a food-reinforced operant task. Seven rats were given repeated non-contingent treatments with morphine (20 mg kg(-1)on saline or no-test days and 10 mg kg(-1)on drug days) starting 20 days before the beginning of discrimination training; another six animals received injections of saline. Chronic administration of morphine resulted in sensitization to the discriminative stimulus effect of this drug and in cross-sensitization to the discriminative stimulus effect of buprenorphine. D-Amphetamine produced only saline lever selection in all rats. In conclusion, the present results confirm that the stimulus properties of opioid drugs may be enhanced, rather than decreased, in animals with a history of repeated non-contingent treatment with morphine. Sensitization to central-acting drugs is thought to play a role in the psychopathology of drug abuse. Hence, the present results point out the necessity of considering the effects of drugs which show tolerance, and those which show sensitization, under any particular drug regimen.     

Mechanism of tolerance development to 2,5-dimethoxy-4-iodoamphetamine in rats: down-regulation of the 5-HT2A, but not 5-HT2C, receptor

  Rationale: Defining the mechanism of tolerance development to hallucinogenic drugs will help to explain their mechanism of action. Objectives: The present study was conducted to determine first, if tolerance develops to the discriminative stimulus (DS) properties of the hallucinogen, 2,5 dimethoxy-4-iodo-amphetamine (DOI) and second, the mechanism mediating tolerance. Methods: Rats were trained to discriminate 0.75 mg/kg DOI from saline on a concurrent VI-30-min schedule of reinforcement with a 15-min time-out for incorrect responses. To evaluate tolerance development, rats were assigned to one of four groups and treated with either chronic saline or chronic DOI. Prior to chronic treatment, two groups were tested for choice behavior following vehicle administration while the remaining two groups were tested following the administration of 0.375 mg/kg DOI. One group from each pre-test condition was injected with either saline or DOI (1 mg/kg) for 8 days. Twenty-four hours after the last chronic injection the pre-test treatments were replicated. Using receptor autoradiography, the density of 5-HT_2A and 5-HT_2C receptors was measured in independent groups of rats that had received identical treatment conditions. Results: Animals receiving chronic DOI showed a 60% decrease in DOI lever responding (from 100% to 40%) when tested on 0.375 mg/kg DOI, while animals receiving chronic saline showed no change in percent choice (100%) on the DOI lever. Significant changes in binding were observed in 5-HT_2A receptors but not 5-HT_2C receptors. The results of tests with antagonists were consistent with the changes in binding. Conclusions: These results suggest that behavioral tolerance to DOI reflects neuroadaptive changes in 5-HT_2A receptors. Received: 17 July 1998 / Final version: 19 January 1999     

Tolerance to the discriminative stimulus and reinforcing effects of ketamine

In order to examine whether tolerance develops to the discriminative stimulus and reinforcing effects of ketamine, rats were trained either to discriminate ketamine (10mg/kg) from saline or to self-administer ketamine (1.1mg/kg/injection), and then treated with chronic ketamine (32mg/kg), administered i.p. every 8 hours for 7 days. No shift in the dose-response curve for either paradigm was obtained following this chronic regimen. However, following a 2-week rest period in which animals had no exposure to ketamine, the dose-response curve was shifted two-fold to the left, indicating increased sensitivity to the drug. Reinstatement of training shifted the dose-response curve back to the right in both paradigms. These results suggest that tolerance to the discriminative stimulus and reinforcing effects of ketamine develops during training. Examination of the self-administration training data support this assumption, since inter-reinforcer time decreases, reflecting an increase in ketamine intake over training sessions.     

Morphine discrimination in the pigeon using a color tracking procedure

Pigeons were trained to discriminate 5.0 mg/kg morphine from saline. After morphine, subjects tracked the location of red response keys and after saline, the location of green keys. When stimulus generalization to other drugs was investigated dl-methadone produced morphine-like responding and this response generalization was primarily due to the l-isomer. Pretreatment with 1.0 mg/kg naloxone shifted the morphine generalization curve 10-fold to the right but only shifted the rate suppression curve 3-fold to the right. dl-Cyclazocine generated dose-related increases in responding on the red key location and in 3 of 5 birds, responses after 1.0 mg/kg were indistinguishable from those after morphine training doses. Meperidine did not produce responding on the red keys, nor did diazepam, cocaine, d-amphetamine, phencyclidine or pentobarbital. The discriminative stimulus effects of morphine are thus stereo-selective and pharmacologically specific. Generalization of responding to dl-cyclazocine but not to phencyclidine suggests that the morphine-like discriminative dl-cyclazocine cue was not due to interaction at sigma opiate receptors.     

Discriminative stimulus properties of d-amphetamine and related compounds in rats

The discriminative stimulus properties of amphetamine were demonstrated in rats trained to discriminate between 0.8 mg/kg of d-amphetamine sulfate and saline. During the discriminative training, animals were shaped on a DRL 15-second schedule to respond to one of two levers for a food reward when they were given d-amphetamine, and to respond to the other lever when they were treated with saline. Tests for the discriminative stimulus properties consisted of 10-min extinction sessions in which the reinforcement delivery was disconnected. Animals receiving low doses (0.2–0.4 mg/kg) of d-amphetamine exhibited mostly saline-like responses, but at a dose of 0.8 mg/kg they produced more than 80% responses on the amphetamine lever. Doses higher than 2.4 mg/kg caused an initial stereotyped behavior and the animals showed a period of latency before responding on the amphetamine lever. In order to elucidate the structural characteristics of d-amphetamine involved in the production of the discriminative stimulus properties, a number of amphetamine derivatives and related compounds were administered to these animals. 1-Amphetamine, ephedrine, norephedrine, 4-methoxyamphetamine and methylphenidate all produced the discriminative stimulus properties similar to d-amphetamine, but doses of 2–10 times greater than d-amphetamine were necessary. Mescaline, STP and DOET did not produce the d-amphetamine-like responses. These results suggest that most psychomotor stimulants, although having different structures, are likely to produce discriminative stimulus properties similar to d-amphetamine.     

Tolerance to the discriminative stimulus effects of Delta(9)-tetrahydrocannabinol

Although tolerance to a variety of behavioral and physiological effects of Delta(9)-tetrahydrocannabinol (Delta(9)-THC) has been demonstrated, previous studies have reported that tolerance to the discriminative stimulus effects of Delta(9)-THC does not develop when discrimination training is continued during repeated administration. The present study investigated development of tolerance to the discriminative stimulus effects of Delta(9)-THC under conditions of supplemental administration during suspended training. Rats, trained to discriminate Delta(9)-THC (3mg/kg) from vehicle in a two-lever drug discrimination procedure, under a fixed-ratio 10 schedule of food reinforcement, were tested with cumulative doses of Delta(9)-THC before and after repeated administration of vehicle and of high doses of Delta(9)-THC. Following suspended training with repeated vehicle injection, the Delta(9)-THC dose-effect curve for percentage of drug lever responding showed little change from the prevehicle curve. After supplemental administration of Delta(9)-THC, the degree of rightward shift in the post-THC dose-effect curve was 40-fold. Recovery to pre-THC levels of percentage of drug lever responding was observed during a second post-THC dose-effect curve administered 23 days later. The large reversible shift in the dose-effect curve following supplemental administration of Delta(9)-THC suggests that tolerance developed to the discriminative stimulus effects of Delta(9)-THC under suspended training conditions.     

Influencing the specificity of drug mixture discriminations by varying the training procedure

Studies of the discriminative stimulus effects of drug mixtures provide an approach to polydrug abuse and to studies on single drugs with multiple effects. The experiments described here investigated whether the use of the AND-OR procedure increases the specificity of drug mixture discriminations. Rats were trained to discriminate a mixture of nicotine (0.4 mg/ kg) plus midazolam (0.2 mg/kg) from saline (AND-discrimination, n = 10) or to discriminate the same mixture from its component drugs alone (AND-OR discrimination, n = 10). The studies used two-lever operant procedures with a tandem variable interval 1 min fixed ratio 10 (FR 10) schedule of food reinforcement. Under AND-discrimination conditions, there was partial generalization to amphetamine and pentobarbitone when each drug was administered singly. With the AND-OR-discrimination, there was no generalization to amphetamine and partial generalization to pentobarbitone. In 'single substitution' tests, pentobarbitone or amphetamine was co-administered with the training doses of nicotine and midazolam, respectively; there was full generalization in the AND-discrimination and no generalization under AND-OR conditions. In 'dual substitution' tests, mixtures of amphetamine plus pentobarbitone produced full generalization under AND-discrimination conditions, and partial generalization in the AND-OR procedure. Wherever comparisons were made, generalization was less under AND-OR- than under the AND-discrimination procedure, confirming that the AND-OR procedure can increase the specificity of discriminations based on drug mixtures. The similarity with findings reported previously for training with mixtures of amphetamine plus pentobarbitone suggests that this may reflect a general principle rather than a phenomenon restricted to particular training drugs.     

Defeat engenders pentylenetetrazole-appropriate responding in rats: antagonism by midazolam

Defeat and the threat of defeat by an aggressive conspecific is stressful and may engender an anxiety-or fear-like state in animals; the present experiment investigated whether defeat generalized to the discriminative stimulus properties of PTZ and how benzodiazepine receptors were involved in this generalization. Separate groups of male Long-Evans rats (Rattus norvegicus) were trained to discriminate 20 mg/kg pentylenetetrazole (PTZ) or 0.4 mg/kg midazolam (MDZ) from saline in a two-choice drug-discrimination task. After establishing stimulus control, PTZ- and MDZ-trained rats were exposed to an aggressive conspecific which resulted in defeat, as defined by the display of defensive and submissive postures as well as audible and ultrasonic vocalizations. Administration of saline after defeat resulted in greater than 80% PTZ lever selection in 15 out of 25 PTZ-trained rats; this effect was attenuated through pretreatment with MDZ (1 mg/kg). Furthermore, short-term defeat substitution for the PTZ discriminative stimulus was not accompanied by long-term changes in the post-defeat generalization curves for PTZ and MDZ when compared to pre-defeat generalization curves. Nor did defeat alter the antagonism of PTZ by diazepam (2.5 mg/kg) or MDZ by flumazenil (10 mg/kg). In order further to characterize the necessary features for defeat substitution for the PTZ discriminative stimulus, exposure to a threatening conspecific was also attempted by PTZ-trained rats protected from physical contact with a wire mesh cage. In these tests, saline continued to engender greater than 50% PTZ lever responding in 15 of 25 rats. These results suggest that an anxiety-like state is induced during defeat and exposure to a threatening conspecific in most rats; this state, as well as the PTZ discriminative stimulus, can be reversed by benzodiazepine receptor agonists. In contrast, short-term defeat substitution for the PTZ discriminative stimulus does not appear to be related to long-term alterations in the benzodiazepine receptor. Present address: Division of Behavioral Biology, Johns Hopkins University School of Medicine, Hopkins Bayview Research Campus, Baltimore, MD 21224, USA     

Nicotine trace discrimination in rats with midazolam as a mediating stimulus

It was shown previously that effects of drugs present prior to training sessions could serve as discriminative stimuli. Further experiments have aimed to determine whether a second drug can serve as a mediating stimulus that increases the strength of stimulus control by such pre-session drug effects. Rats were trained in a two-lever discrimination procedure with food reinforcers presented on a tandem variable-interval fixed-ratio (VI-FR) schedule. Injections of nicotine (0.6 mg/kg) or saline were followed after 5 min by administration of midazolam (0.2 mg/kg) as a putative mediating stimulus. The nicotine antagonist mecamylamine (1.0 mg/kg) was administered 5 min after midazolam, to block effects of nicotine during training sessions, as in previous work on pre-session drug effects. Stimulus control was acquired slowly and to an accuracy of only 75%. Midazolam did not facilitate the acquisition or magnitude of nicotine-induced stimulus control. However, extinction tests showed that the presence of midazolam was required for expression of stimulus control by pre-session effects of the training dose of nicotine. The response to nicotine (0.075-0.6 mg/kg) was dose-related, but the dose-response relationship was not dependent upon the presence of midazolam. In a group of rats trained with nicotine and midazolam as above, but without mecamylamine, stimulus control by nicotine was not dependent upon the presence of midazolam. In all cases, overall rates of responding were very low when tests were carried out without midazolam, suggesting the presence of state-dependent learning. The results imply that under appropriate conditions the discriminative stimulus effects of one drug (nicotine) can be mediated by the action of a second substance (midazolam). This finding can be conceptualized in terms of occasion setting, with nicotine serving as the feature and midazolam as the target stimulus. Furthermore, it appears that even when rates of responding show drug-state dependence, this is not necessarily the case for discriminative stimulus effects.     

Discriminative stimulus properties of fentanyl and morphine: tolerance and dependence.

Using a food-reinforced two-lever operant procedure, rats were trained to discriminate 0.04 mg/kg fentanyl from saline. At different time intervals after the establishment of discriminative responding, stimulus generalization experiments were performed with equivalent dose ranges of fentanyl (0.0025 to 0.02 mg/kg) and morphine (2.5 to 20 mg/kg). It was found that the ED50 values of both compounds for generalization with the narcotic discriminative stimulus complex, did not change over a 4-month period. The subjects used in this study demonstrated marked tolerance to narcotic analgesia, but none of them showed signs reminescent of narcotic dependence. It is concluded that the discriminative stimulus properties of narcotic analgesics are not subject to tolerance.     

Evidence for dopaminergic involvement in tolerance to the discriminative stimulus properties of cocaine

Rats were trained to discriminate cocaine, 10.0 mg/kg, in a two-lever operant procedure. Dose-effect data for generalization to cocaine and substitution of apomorphine for the cocaine stimulus were determined. Subsequently, training was halted and either apomorphine, 2.5 mg/kg per 8 h, or cocaine, 20 mg/kg per 8 h, was administered for 7-9 days. During chronic administration, the efficacy of cocaine and apomorphine as discriminative stimuli was decreased. These data suggest that dopaminergic mechanisms may mediate tolerance to the discriminative stimulus properties of cocaine.     

Discriminative stimulus effects of melatonin in the rat

To provide initial information on the potential mechanisms underlying the discriminative stimulus effects of melatonin, rats were trained to discriminate melatonin (150 mg/kg, IP) from saline in a two-choice discrete-trial avoidance paradigm. Stimulus generalization curves for melatonin were steep; complete generalization with melatonin occurred at 100–150 mg/kg. Triazolam generalized completely with melatonin (n=7). Flurazepam generalized completely with melatonin in only two out of six rats; however, partial generalization was produced in the remaining four animals. The melatonin-appropriate responding produced by triazolam was antagonized completely (in six out of seven rats) by 0.3–10 mg/kg flumazenil (Ro 15–1788). In contrast, the dose of flumazenil sufficient to block completely the melatonin-like discriminative effects of triazolam failed to block the stimulus effects of the training dose of melatonin. Pentobarbital produced primarily melatonin-appropriate responding, with complete generalization with melatonin in five out of seven rats. Diphenhydramine generalized completely with melatonin in two out of seven rats; however, little or no partial generalization was observed in the remaining five rats. These results suggest that melatonin may produce its discriminative effects through sites on the GABA_A-benzodiazepine receptor complex distinct from the benzodiazepine binding sites.     

Serotonergic-dopaminergic mediation of MDMA's discriminative stimulus effects in a three-choice discrimination

(+/-)3,4-Methylenedioxymethamphetamine (MDMA; "Ecstasy") is a common drug of abuse that is often described as both a psychostimulant and a hallucinogen. Two-choice drug discriminations (i.e. drug vs. nondrug) in nonhumans comparing the discriminative stimulus properties of MDMA to psychostimulants or hallucinogens have produced somewhat inconsistent findings. The relative contribution of serotonergic versus dopaminergic actions to MDMA's discriminative stimulus effects may depend on the training stimulus conditions employed. We have previously demonstrated that rats can learn to discriminate the effects of MDMA and D-amphetamine in a three-choice drug discrimination procedure, and that LSD produced nearly complete substitution for MDMA under these conditions, and fenfluramine fully substituted for MDMA. In the present study, 12 rats were trained to discriminate LSD (0.08 mg/kg) and MDMA (1.5 mg/kg) from saline in a three-choice drug discrimination procedure under a fixed-ratio (FR) 10 schedule of food reinforcement. D-Amphetamine produced only partial substitution for MDMA while fenfluramine produced complete stimulus generalization. Low doses of D-amphetamine and fenfluramine produced greater stimulus generalization when administered in combination than when given alone. The serontonin(2) antagonist MDL-100,907 only partially blocked the MDMA cue, but completely antagonized LSD discrimination. The dopamine antagonist haloperidol also failed to block MDMA discrimination. These results indicate that 5-HT release is a salient feature to MDMA's discriminative stimulus effects but that MDMA produces a compound discriminative stimulus.     

Tolerance to morphine stimulus control: role of morphine maintenance dose

Experiments assessed the development of tolerance to morphine stimulus control during treatment with selected maintenance doses of morphine. Separate groups of rats were trained to discriminate saline and either 3.2 mg/kg or 5.6 mg/kg morphine under fixed-ratio schedules of food delivery. Dose-response functions for generalization of morphine stimulus control were determined before, during, and after repeated treatment with selected doses of morphine. Similar experiments were performed with repeated pentobarbital treatment in order to assess the pharmacological selectivity of tolerance. Repeated treatment with saline, 3.2 mg/kg morphine, or twice daily injections of 17.8 mg/kg pentobarbital produced no tolerance to morphine stimulus control. In contrast, treatment with daily injections of 10 mg/kg or twice daily injections of 10 or 17.8 mg/kg morphine produced a dose-dependent increase in the dose of morphine required for stimulus control. The magnitude of tolerance to morphine stimulus control varied directly with the maintenance dose of morphine and was slightly greater for a lower than a higher morphine training dose. Termination of repeated treatment was followed by a return to initial sensitivity, without additional training. Tolerance to morphine stimulus control was not necessarily accompanied by tolerance to its rate-suppressing effects.     

Impact of training history on discrimination of a drug mixture by rats

The impact of training sequence on discrimination of a mixture of two drugs was investigated with five groups of rats (n = 10). In phase I, two groups were trained according to conventional two-lever, operant drug discrimination protocols with food reinforcement; one of these groups was trained with nicotine (0.4mg/kg) and the other group was trained with midazolam (0.15mg/kg). The three remaining groups served as controls and were subjected to 'sham' training in which administrations of saline, nicotine or midazolam were unrelated to contingencies of reinforcement. After completion of phase I (40 sessions), all five groups were trained to discriminate a mixture of nicotine (0.4mg/kg) plus midazolam (0.15mg/kg) from saline (phase II). Any differences between the groups in their performance during phase II could, therefore, be attributed to their different histories in phase I. During phase II, all groups discriminated the mixture from saline with similar accuracy (89-94% drug-appropriate responding after mixture as compared with 2-7% after saline). In the three groups of rats subjected to 'sham' training in phase I, there was partial generalization to both nicotine (45-53%) and midazolam (39-40%), each of which therefore contributed about equally to stimulus control by the mixture. In rats that were initially trained to discriminate nicotine, midazolam had acquired little stimulus control over behaviour (9%) and discrimination of the mixture was attributable largely to the nicotine (87%). Conversely, in rats that were initially trained to discriminate midazolam, nicotine contributed 3% and midazolam 76% to stimulus control by the mixture. These powerful, persistent effects of training sequence were interpreted as examples of associative blocking demonstrated with the interoceptive stimuli produced by psychoactive drugs.